Development and application of a scalable workflow for immunomagnetic separation of exRNA carrier subclasses and molecular analysis of their cargo

摘要 在所有被测试的人体体液中都发现了细胞外RNA(ExRNAs)，而且有越来越多的证据表明 它们可以作为细胞间沟通的媒介，以及诊断、预后和 广泛的疾病和生理条件的鼻音生物标志物。ExRNAs与 各种载体亚类(CS)，包括细胞外小泡(EVS)、核糖核蛋白复合体(RNP)、 以及脂蛋白(LPP)，其中许多迄今尚不清楚或特征不佳。这个跨学科的团队， 在exRNA和脂蛋白生物学、exRNA生物标记物发现、exRNA治疗、exRNA 测序、低投入蛋白质组和脂质组学以及流式细胞术将共同开发和应用一种 严格、可重复、高效、可扩展且经济实惠的免疫磁分离(IMS)工作流 用于下游基因组分析的CS的制备性分离。此外，基于珠的多路传输的潜力 我们将探讨如何通过流排序来实现更高效的CSS分离。目标1A将专注于开发 使用适当的细胞培养模型鉴定和分离已知和可疑的CS的试剂 以及健康的人体血浆和血清样本。这项工作将包括筛选可用的抗体。 已知的一般CS(如Tetraspanins、AGO蛋白、载脂蛋白)和各种细胞类型的标记- 识别在Western Blot和IMS中表现良好的抗体的特定标记，以及结果的传播 成功的和不成功的抗体。在目标1B中，目标1A的结果将应用于建造和 测试IMS工作流程，以分离和分析一般的RNAseq、蛋白质组和脂组 Css和特定于细胞类型的电动汽车。除了构建包含RNA的全面知识集之外， 已知和可疑的CS的蛋白质和脂类货物，分析未被IMS捕获的材料 工作流程将展示新颖的css。目标2A将包括将IMS工作流程和应用程序细化到三个 临床队列：怀孕和未怀孕的女性对照组；心肌梗死后和年龄-性别- 匹配的高危对照组；上皮性卵巢癌和年龄匹配的健康女性对照组。分析 通过小RNAseq和长RNAseq与一般的CSS和细胞类型特定的EV相关的exRNAs将揭示 这些css的相对丰度和exRNA含量在这些病例和对照中是否有所不同 一群人。目标2B将包括开发一种基于流式细胞术的多路同时传输策略 人血浆和血清中多组分的分离。如果成功，这个项目将导致开发 严格的工作流程，用于分离可重复且快速地产生的 在临床上以经济高效的方式高度浓缩所需的CS，并最大限度地减少其他CS的污染 可行的进样体积，并产生足够的下游分子分析材料。此外, 在该项目过程中产生的全面的基因组数据将产生有价值的参考侧写 关于以前已知的和新的CS携带的RNA、蛋白质和脂肪货物的数据。

SUMMARY Extracellular RNAs (exRNAs) have been found in all tested human biofluids, and there is increasing evidence that they can serve as mediators of intercellular communication, as well as diagnostic, prognostic, and theranostic biomarkers for a wide range of disease and physiological conditions. ExRNAs are associated with a variety of carriers subclasses (CSs), including extracellular vesicles (EVs), ribonucleoprotein complexes (RNPs), and lipoproteins (LPP), many of which are as-yet unknown or poorly characterized. This transdisciplinary team, with expertise in exRNA and lipoprotein biology, exRNA biomarker discovery, exRNA therapeutics, exRNA sequencing, low-input proteomics and lipidomics, and flow cytometry, will work together to develop and apply a rigorous, reproducible, efficient, scalable, and cost-effective immunomagnetic separation (IMS) workflow for preparative isolation of CSs for downstream omic analysis. In addition, the potential for multiplex bead-based flow sorting for even more efficient separation of CSs will be explored. Aim 1A will focus on development of reagents for identification and separation of known and suspected CSs using appropriate cell culture models and healthy human plasma and serum samples. This work will include screening of available antibodies against markers for known general CSs (e.g. tetraspanins, AGO proteins, apolipoproteins) and a variety of cell type- specific markers to identify antibodies that perform well for Western Blot and IMS, and dissemination of results for both successful and unsuccessful antibodies. In Aim 1B, the results from Aim 1A will be applied to build and test an IMS workflow for separation and small and long RNAseq, proteomic, and lipidomic analysis of general CSs and cell type-specific EVs. In addition to building a comprehensive knowledge set encompassing the RNA, protein, and lipid cargo of known and suspected CSs, profiling the material that is not captured by the IMS workflow will reveal novel CSs. Aim 2A will encompass refinement of the IMS workflow and application to three clinical cohorts: Pregnant and non-pregnant female controls; Post-myocardial infarction and age- and sex- matched at-risk controls; and Epithelial ovarian cancer and age-matched healthy female controls. Analysis of the exRNAs associated with general CSs and cell type-specific EVs by small and long RNAseq will reveal whether the relative abundance and exRNA cargo of of these CSs differs between cases and controls in these cohorts. Aim 2B will consist of development of a flow cytometry-based strategy for multiplexed simultaneous separation of multiple CSs from human plasma and serum. If successful, this project will result in development of a rigorous workflow for separation of exRNA CSs that reproducibly and rapidly produces fractions that are highly enriched for desired CSs with minimal contamination by other CSs in a cost-effective manner on clinically feasible volumes of input material, and yields sufficient material for downstream molecular analysis. In addition, the comprehensive omic data generated on during the course of this project will yield valuable reference profiling data on the RNA, protein, and lipid cargo carried by previously known and novel CSs.